P. berghei malaria can successfully be induced in mice by immunization with both radiation attenuated sporozoites (RAS) arresting early during liver stage development, or sporozoites combined with chloroquine chemoprophylaxis (CPS), resulting in complete intra-hepatic parasite development before killing of blood-stages by chloroquine takes place. We assessed the longevity of protective cellular immune responses by RAS and CPS P. berghei immunization of C57BL/6j mice. Strong effector and memory (TEM) CD8+ T cell responses were induced predominantly in the liver of both RAS and CPS immunized mice while CD4+ T cells with memory phenotype remained at base line levels. Compared to unprotected naïve mice, we found high sporozoite-specific IFNγ ex vivo responses that associated with induced levels of in vivo CD8+ TEM cells in the liver but not spleen. Long term evaluation over a period of 9 months showed a decline of malaria-specific IFNγ responses in RAS and CPS mice that significantly correlated with loss of protection . The reducing IFNγ response by hepatic memory CD8+ T cells could be boosted by re-exposure to wild-type sporozoites. Our data show that sustainable protection against malaria associates with distinct intra-hepatic immune responses characterized by strong IFNγ producing CD8+ memory T cells.Protection against  Plasmodium infected mosquitoes that inject sporozoites into the skin. These sporozoites travel to the liver for further development and released as blood-stage parasites that are responsible for clinical malaria Malaria is transmitted to the host through bites of P. berghei sporozoites.Sterile protection against malaria by whole sporozoites is thought to be mediated by hepatic CD8+ T cell responses. The expansion of CD8+ T cells with memory phenotype, identified by the high expression of CD44, as well as high production of IFNγ have been shown to associate with protection by RAS P. berghei ANKA sporozoites (Pbspz) according to RAS or CPS protocols (EM: CD44hiCD62L−) and without obvious alterations in the central memory (TCM: CD44hiCD62L+) pool  or low (10 K/10 K/10 K) dose of rotocols . Inducedrotocols . Both RAL+) pool . In spleBoth high (50 K/20 K/20 K) and low (10 K/10 K/10 K) immunization doses with either RAS or CPS regimes induced complete protection in 100% of the mice . All naïEM cells. Prior to challenge (C-1) the proportion of CD8+ TEM cells was higher in PBMC, HMC and splenocytes of RAS-compared to CPS immunized mice  while the post-immunization profile remained stable in CPS immunized mice. Overall, CD8+ but not CD4+ TEM cells, only from liver but not spleen or peripheral blood remained significantly higher in immunized versus naïve mice. Interestingly, CD8+ TEM cells from naïve mice significantly increased during fatal infection up to day 6 post-challenge (p = 0.008), most strikingly in the liver but also in spleen and peripheral blood. All data combined indicate the strongest memory T cell responses to be generated intra-hepatically.We next studied the effect of challenge infections on the dynamics of CD4+ and CD8+ Tzed mice . There wex vivo stimulated with cryo-conserved P. berghei sporozoites. CD8+CD44hi T cells of RAS and CPS mice show similar IFNγ responses albeit somewhat higher in the liver than in the spleen (ex vivo IFNγ response to Pbspz while the positive control stimulation with PMA and ionomycin resulted in percentage of responding cells similar to immune mice (data not shown).IFNγ responses of CD8+ T cells with memory phenotype (CD44hi) induced by RAS or CPS immunization was assessed 21 days following challenge at day 41 (C+21). Freshly isolated HMC and splenocytes were e spleen . Liver aEM were assessed 3, 6 or 9 months following the last immunization by three doses of 10 K sporozoites. Analysis of the memory pool showed compared to naïve mice, sustained and significantly high levels of CD8+ T cells with effector memory phenotype up to 9 months post- immunization  or infected red blood cells (PbiRBC) were significantly increased, with the highest responses in RAS immunized mice(Pbspz specific IFNγ response was observed over a 3 to 9 months post-immunization period (We further explored the differences in antigen exposure of RAS and CPS protocols (tested by serology) and assessed the longevity of IFNγ response by hepatic CD8+ T cells. While concentrations of anti-sporozoite specific IgG antibodies were equal in all immunized mice, only the CPS protocol as expected induced anti-blood-stage specific IgG antibodies . Challenized mice. Howevern period . In cont2 = 0.60, p<0.0001).Long-term protection was evaluated by challenge at 3, 6 or 9 months post immunization. While RAS protocol induced 100% protection at all time points, CPS-induced protection was reduced from 100% (t = 3 and 6 months) to 50% (t = 9 months) . InteresEM cell response could be boosted by re-exposure. In protected mice challenged 3, 6 or 9 months after immunization, levels of liver CD8+ TEM showed at day 21 after individual challenge infections a significant increase in proportion of CD8+ TEM .We finally investigated whether decreased liver CD8+ TCD8+ TEM . MoreoveCD8+ TEM . AlthougP. berghei RAS and CPS immunization, similar T cell responses are induced with increase of predominantly the CD8+ T cell pool with memory phenotype in liver, and to a lesser extent in spleen and PBMC. The composition of the CD4+CD44hi T cell pool remains relatively unaffected. The observed changes after RAS immunization corroborate data from previous studies showing clear liver CD8+ TEM cells responses with modest expansion of spleen cells and PBMC EM cells with IFNγ as one of the main actors. Further long term evaluation of RAS or CPS induced immune responses and protection clearly shows that up to 9 months after immunization, malaria specific IFNγ response declines despite sustained high levels of liver CD8+ TEM cells. This waning IFNγ response significantly correlates with loss of protection observed in CPS mice 9 months after immunization . In a previous study by Jobe et al P. berghei RAS immunization was shown to induce liver IFNγ-secreting CD8+ T cells still measurable at high levels upon re-challenge 6 months after the first challenge infection. While these findings are in line with our post-challenge observations, our pre-challenge data show that in its natural course, CD8+ T-cell mediated protective immune response declines over time yet remains detectable months after immunization prior to boost by re-exposure.The present study highlights the essential role of IFNγ response by liver memory CD8+ T cell for the longevity of protection against malaria. After both loCD11ahi T cells in peripheral blood loCD11ahi T cells were however not sufficient to induce protection. Similar findings were reported by Friesen et al, showing equally high levels of PBMC CD8aloCD11ahi T cells in mice immunized by P. berghei RAS or under azythromycin chemoprophylaxis despite protection levels of 40 and 80% respectively loCD11ahi T cells (as shown by others) or CD8+ T cells with classical effector memory phenotype (CD44hiCD62L−) are sufficient to predict protection. On top of quantitative analysis of CD8+ memory T cell responses, the present study supports the predictive value of malaria specific IFNγ response for longevity of protective immunity.Several studies have addressed protection in murine malaria models with parallel immune responses. Schmidt et al have shown induced and sustained (up to 5 months) CD8+ memory T cell response defined by presence of CD8aP. yoelii parasites induce a more robust protective immune response in mice compared to RAS P. berghei and P. yoeliiOur findings of a more sustained protective immunity induced by RAS compared to CPS are somewhat surprising. As shown by Butler et al., late arresting genetically attenuated ‘central mediator of protective immune response against malaria’Finally, encouraging long term protection up to 42 ad libitum. All animal studies and procedures were approved by the Ethical Committee on Animal Research of the Radboud University Nijmegen .C57BL/6j mice (6 to 8 weeks old) were purchased from Elevage-Janvier . Mice were housed at the Central Animal Facility in Nijmegen and received a standard diet and water P. berghei (ANKA) sporozoites (Pbspz) were obtained by dissection of the salivary glands of infected female Anopheles stephensi mosquitoes 21–29 days after blood meal on infected mice. To obtain radiation attenuated sporozoites (RAS), infected mosquitoes were irradiated at 16,000 rad (Gammacell 1000 137Cs) prior to dissection.Pbspz doses: 50.000-20.000-20.000 or 10.000-10.000-10.000. During CPS immunization, mice received a daily i.p. injection of 800 µg of chloroquine base starting simultaneously with the first inoculation up to two weeks after the last sporozoite inoculation. Chloroquine disphosphate  was diluted in PBS and administered to both immunized and naïve mice. At the end of the chloroquine treatment and approximately a day before challenge, absence of parasitemia was confirmed by examination of Giemsa-stained slides of tail blood.Mice received three intravenous injections with weekly intervals of two Pbspz. In one experiment, mice were challenged by bites of 5–11 infected mosquitoes. Presence of sporozoites in mosquitoes after feeding was confirmed by examination of the dissected salivary glands.Absence of blood-stage parasites prior to challenge was confirmed at the end of the rested phase in all groups. CPS or RAS immunized and naïve mice were simultaneously challenged at day 41 or 3 to 9 months after the last immunization by i.v. injection of 10,000 WT Parasitized red blood cells were identified by Giemsa-stained blood smears on other days from day 3–14 and finally on day 21 after challenge. Protection was defined as the absence of blood-stage parasites by day 21 post-challenge. A schematic representation of the experimental design is presented in P. berghei sporozoites or infected red blood cells (iRBC) was performed as described by Bousema et al g RT)and overnight coating of Sterilin ELISA plates  was performed with the equivalent of 40.000 iRBC or 4.000 sporozoites per well. After blocking with 5% milk/PBS, plasma samples were incubated for three hours at room temperature . Rabbit anti-mouse IgG HRP  was used for the detection of IgG antibodies to sporozoites or blood-stage antigens.Antigen extraction from whole Before and after challenge, mice were euthanized by isoflurane inhalation after i.v. injection of 50 IU of heparin. Blood, spleen and liver were collected after perfusion of the liver with 10 ml of PBS. Cell suspensions of liver and spleen were made by passage of the organs through a 70-µm nylon cell strainer (BD Labware). Liver cells were resuspended in 35% Percoll  and centrifuged at 800 g for 20 min. Liver and spleen erythrocytes were lysed by 5 min incubation on ice in ACK lysing solution. After erythrocyte lysis, HMC and splenocytes were resuspended in PRMI 1640 medium. Isolation of peripheral blood mononuclear cells (PBMC) was performed using Histopaque-1077 (Sigma-Aldrich) according to the manufacturer's recommendation.6 cells were resuspended in cold assay buffer  and incubated for 30 min at 4°C with the monoclonal antibodies. Cells were fixed with Fix & Perm medium A (Invitrogen) and resuspended in assay buffer for measurement.Five-color staining of PBMC, HMC and splenocytes was performed using monoclonal antibodies purchased by Biolegend: Pacific blue-conjugated anti CD3 (17A2), Peridinin Chlorophyll Protein (PerCP)-conjugated anti CD4 (RM4.5), Alexa fluor 700-conjugated anti CD8a (53–6.7), fluorescein isothiocyanate (FITC)–conjugated anti-CD44, allophycocyanin (APC)– or phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-CD62L (MEL-14). Briefly, 105 cells/well) were co-cultured in complete RPMI 1640 culture medium 4/ml), P. berghei infected red blood cells (iRBC - 5×106/ml), salivary gland preparations from uninfected mosquitoes or uninfected red blood cells (uRBC - 5×106/ml). Cells were stimulated at 37°C/5%CO2 for 24 hours and Brefeldin A (Sigma) was added during the last four hours . As positive control, PMA (100 ng/ml) and Ionomycin (1.25 µg/ml) (Sigma) were added simultaneously with Brefeldin A. Cells were harvested after 24-hours in vitro stimulation and stained with monoclonal antibodies against CD3, CD4, CD8a and CD44 as indicated above. Fixed cells were stained with APC-conjugated anti-IFNγ with Fix & Perm medium B (Invitrogen) at 4°C for 30 min.HMC and splenocytes . p<0.05 was considered statistically significant.Flow cytometry was performed on a 9-color Cyan ADP (Beckman Coulter) and data analysis using FlowJo software . For the analysis of cytokine production, background response to salivary glands or uRBC was subtracted from


The proportion of mothers who exclusively breastfeed their babies up to 6 months remains low. Determinants of breastfeeding practices have been largely documented in high-income countries. Little evidence exists on possible predictors of breastfeeding behaviors in the Middle East. Our aim was to assess the prevalence of breastfeeding in Beirut and determine the factors that impact breastfeeding behavior in this population.Data for this longitudinal study is nested within a randomized controlled trial (RCT) assessing the impact of a 24-hour hotline and postpartum support film on postpartum stress. Healthy first-time mothers delivering in the capital Beirut between March and July 2009, were interviewed at 1–3 days and 8–12 weeks post delivery. A multiple logistic regression analysis was used to determine the factors associated with exclusive breastfeeding at 8–12 weeks postpartum.The overall breastfeeding rate at 8–12 weeks postpartum was 67%. The exclusive breastfeeding rate was 27.4%. Factors associated with exclusive breastfeeding included maternal work , planned pregnancy , intention to breastfeed , source of maternal emotional support  and the use the postpartum support video, the hotline service or both .The proportion of healthy first-time mothers who exclusively breastfeed in Beirut is extremely low. Factors associated with breastfeeding behavior are diverse. Future research and interventions should target different levels of the maternal-child pair’s ecosystem.NCT00857051ClinicalTrials.gov,  The health benefits of breastfeeding have been widely cited. Breastfeeding has been shown to protect infants against common acute childhood infections, enhance the immune system, decrease rates of Sudden Infant Death Syndrome (SIDS), promote cognitive development and prevent chronic diseases such as obesity, diabetes mellitus type 1 and 2), asthma and certain pediatric malignancies  and 2, a, notwithFor optimal growth and development, the World Health Organization (WHO) recommends exclusive breastfeeding for the first six months of life, starting in the first half hour after delivery . HoweverUnderstanding the context-specific patterns and determinants of breastfeeding practices is necessary to ensure successful breastfeeding promotion strategies . As suchAlthough various breastfeeding promotion initiatives have been established in Lebanon, breastfeeding rates remain low. According to the 2006 Pan Arab Family Health Survey (PAPFAM), while up to 89% of infants are ever breastfed, only 24% of infants below 4 months are exclusively breastfed. The average duration of breastfeeding is 9 months . One stuTo date, studies that have addressed possible predictors of breastfeeding behavior in Lebanon remain scarce. This paper aims to assess the prevalence of breastfeeding in Beirut and determine the factors that impact breastfeeding behavior in this population. A better understanding of these factors would contribute to a larger academic understanding of the breastfeeding ecosystem in Lebanon, assist breastfeeding promotion agencies and practitioners to implement successful strategies, and catalyze the development of effective breastfeeding-friendly policies.Lebanon is a small middle-income country on the Eastern Mediterranean, with an estimated population of 4 million and a fertility rate of 1.7 . Its popThis was a secondary analysis of data from a randomized control trial (RCT) assessing the impact of a 24-hour hotline service and postpartum support film on postpartum stress among first-time mothers [unpublished data - Osman]. First-time mothers were randomized according to a computer-generated random list into one of four groups . A randomized controlled single-blind design was used. The postpartum support film was recorded on a DVD, the hotline service number was marked on a card and the control group entailed a music CD. All materials were placed in a hard DVD cover and in consecutively-numbered opaque envelopes that looked and felt the same. These were handed to every mother by recruiters who were blinded to their contents.Based on power calculations for the original RCT (see below), a total of 751 primiparous women were contacted: 119 were excluded and 80 refused to participate. 552 women received the baseline interview.The study was conducted between March and July 2009. Trained interviewers conducted a baseline and postpartum assessment of healthy first-time mothers at 1–3 days and 8–12 weeks post-delivery respectively.Baseline data was collected daily over a 7-week period by 8 recruiters who included midwives and public health graduate students. Recruiters visited participating hospitals daily at same time (8-10am) to include all primipara deliveries that met study inclusion criteria. At each hospital visit, the recruiter reviewed the list of deliveries in the last 24 hours and visited every woman who met the inclusion criteria in her room. Written informed consent was obtained from each prospective participant. A 3-minute baseline interview was conducted to gather information about the woman’s socio-demographic status, her health and that of her baby as well as her pregnancy, delivery experience and intent to breastfeed.Fourteen assessors were trained to conduct the postpartum interview. Interviews were conducted face to face at women’s homes when possible. Telephone assessments were conducted when assessors were unable to visit women at home. The postpartum interview lasted between 30 to 50 minutes. It included questions about general health, infant health and care, breastfeeding attitude and behavior, marital life, employment and perceptions of self-efficacy and social support. In addition, screening tests for postpartum anxiety and stress as well as postpartum depression were conducted.All first time mothers delivering within participating hospitals were eligible for inclusion in the study. They were excluded if they had any of the following characteristics: 1) multiple or complicated gestations, 2) chronic diseases requiring daily management such as cardiovascular diseases, hypertension, diabetes, or thyroid diseases, 3) infant in the neonatal intensive care unit, or 4) they were planning on leaving Lebanon before the time of assessment.The outcome of this study was exclusive breastfeeding at 8 to 12 weeks postpartum. It was categorized into exclusive breastfeeding and non-exclusive breastfeeding (consisting of complementary feeding and infant milk formula). As per the WHO definition, exclusive breastfeeding was defined as giving no other food or drink, not even water, except breast milk (including milk expressed or from a wet nurse) but allows the infant to receive oral rehydration solution (ORS), drops and syrups .Based on a thorough literature review, a set of factors was selected to assess their associations with the outcome Table . These iSample size was calculated based on the aim of reducing the PSS-10 mean by 4 points in the original RCT. The mean score for the PSS-10 was found to be 18.3, with a standard deviation of 4.9 in the validation study among postpartum women in Lebanon. Based on the assumption that 50% would watch the film, the mean for the intervention group was considered to be 16.3. Therefore, 126 women were needed in each arm with an alpha of 0.05 and a power of 90%. Accounting for 10% loss to follow-up, 140 women were needed for each arm. Although these power calculations were conducted for the original RCT, they are similarly adequate for this cross-sectional study given the multiple outcomes tested and allowed for.The statistical package for social sciences (SPSS) version 16 was used. Descriptive statistics were done to examine variability of all chosen factors and to decide on bracketing  on March 5, 2009. Applications were also submitted and approved by ethical review boards of participating hospitals when required.A total of 751 primiparous women were approached to participate with a 74% enrollment rate (119 were excluded and 80 refused to participate). There were no significant differences between the socio-demographic characteristics of women who participated and those who refused (data not shown). Of the 552 women who received the baseline assessment, 452 (82%) completed the postpartum questionnaire and 100 (18%) were lost to follow-up. Of the 452 who completed the postpartum assessment, 325 (72%) had a face-to-face interview and 127 (28%) completed it over the telephone. There were no significant differences between the socio-demographic characteristics of women who were assessed and those who were lost to follow up.The majority of women had secondary education and above (70.8%), with slightly over half of them 51.2%) having reached university. Around sixty percent were unemployed and reported a household monthly income of over 1 million Lebanese pounds . Most women did not report any health problem in the postpartum period. However, the prevalence of stress, anxiety and depression symptoms was relatively high . The large majority of newborns was term (91.4%) and close to half (46.3%) was delivered by C-section. The largest percent of mothers intended to exclusively breastfeed (87.1%) (Table % having Most (67%) mothers were breastfeeding at 8–12 weeks postpartum. Only 27.4% of mothers were exclusively breastfeeding at that time, 39.6% were giving both breast milk and infant formula while 33% were giving infant formula only.p-value= 0.092 and 0.068, respectively). Factors significantly associated with exclusive breastfeeding included maternal age, employment and monthly household income, gestational age and mode of delivery, intention to breastfeed at the time of delivery, baby’s health and main source of emotional support for the new mother.Various factors were tested for their association with exclusive breastfeeding Table . These wp-value= 0.003) compared with 30.4% of younger mothers. Only 13.8% of working mothers exclusively breastfed compared with 35.8% of non-working mothers . Interestingly, 18.2% of those whose monthly household income was more than 2 million Lebanese Pounds  exclusively breastfed their infant compared with 33.3% of those whose monthly income was below 1 million Lebanese Pounds (around 600 US Dollars) . Only 11.1% of mothers of preterm infants exclusively breastfed compared with 27.1% of mothers of term infants . 22.2% of those who underwent a C-section exclusively breastfed compared with 32.5% of those who underwent a vaginal delivery .41.2% of mothers whose ages were between 20 and 24 exclusively breastfed their infant . 30% of women who intended to exclusively breastfeed at the time of delivery were doing so, compared to 6.9% of those who did not intend to. A significantly higher proportion of mothers who did not report any health problems in their newborn breastfed exclusively compared to those who reported that their babies had health problems .Intention to exclusively breastfeed was strongly associated with exclusive breastfeeding .21.1% of women who identified their mothers as their main source of emotional support exclusively breastfed compared with 31.8% of women who reported their husbands or others as their primary source of emotional support (Maternal work, intention to breastfeed, pregnancy planning, source of emotional support and the intervention arms were significantly associated with exclusive breastfeeding Table . Non-worThis study confirms the low prevalence of exclusive breastfeeding at 8–12 weeks postpartum in a representative sample of women in Beirut. The large majority of the mothers in this study were not exclusively breastfeeding at 8–12 weeks postpartum. Prevalence of exclusive breastfeeding at 6 months postpartum is likely to be even lower. This finding is similar to findings from other developing countries demonstrating the low adherence to the WHO breastfeeding guidelines ,27. In LIn this study, almost all mothers intended to exclusively breastfeed, similar to observations in neighboring countries such as Syria and Jordan . The facThe large C-section rate found in this study was similar to C-section rates in Beirut reported in other studies -33. AlthThe hotline service instituted as part of the original RCT also had a significant impact on exclusive breastfeeding. Women who used the hotline were almost four times as likely to exclusively breastfeed. This may indicate the need for more awareness and/or support to breastfeeding mothers. In today’s ever-growing time constraints, our result emphasizes the value, for women’s health practitioners, of simply taking the time to answer a question. Given that the hotline issues addressed were not restricted to breastfeeding, it would be interesting to investigate whether the impact of a “breastfeeding hotline” would be greater. If so, a national hotline could prove to be a cheap and feasible way to raise breastfeeding rates.The restriction of participants to healthy first-time mothers delivering in Beirut is one limitation of this study. The conclusions reached may not be applicable to the larger Lebanese population, especially given the likely differences in breastfeeding practices among urban and rural mothers . FurtherThe proportion of healthy first-time mothers who exclusively breastfeed in Beirut is extremely low. This evidence is a timely reminder that much has yet to be done to promote breastfeeding in Lebanon. Understanding its determinants is a first step towards developing successful interventions and policies. This study has found that maternal employment status, intent to breastfeed and baby’s gestational age and physical health are significantly associated with exclusive breastfeeding. The diversity of these factors dictates an ecological approach to breastfeeding promotion. Multi-faceted interventions across different levels of the maternal-child pair’s ecosystem are likely to be the most effective. Beyond the epidemiology of breastfeeding, further research is needed to assess which multi-level strategies most effectively raise breastfeeding outcomes.The authors declare that they have no competing interests.HH contributed to the literature review and interpretation of data. MC contributed to the design, analysis and interpretation of data. MS contributed to data collection, management and analysis. RC contributed to the literature review and data analysis. HO contributed to the conceptualization of the study design, analysis and interpretation of data. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/702/prepub


The past two years have witnessed unprecedentedly rapid development of organic–inorganic halide perovskite–based solar cells. The solution–processability and high efficiency make this technology extraordinarily attractive. The intensive investigations have accumulated rich experiences in the perovskite fabrication; while the mechanism of the chemical synthesis still remains unresolved. Here, we set up the chemical equation of the synthesis and elucidate the reactions from both thermodynamic and kinetic perspectives. Our study shows that gaseous products thermodynamically favour the reaction, while the activation energy and “collision” probability synergistically determine the reaction rate. These understandings enable us to finely tune the crystal size for high-quality perovskite film, leading to a record fill factor among similar device structures in the literature. This investigation provides a general strategy to explore the mechanism of perovskite synthesis and benefits the fabrication of high–efficiency perovskite photoactive layer. The recent exploitation of organic–inorganic hybrid perovskite in solar energy conversion arouses new academic curiosity1358AMX3, where A is methylamine, M is metal element and X represents halide element1112132 (M = Pb or Sn) and CH3NH3X (X = Br or I) with a molar ratio of 1:3. The preparation of CH3NH3PbIx3-Clx film through reaction (1) is a typical example2  and CH3NH3X (X = Br or I) with a molar ratio of 1:1, such as the reaction between PbI2 and CH3NH3I for the synthesis of CH3NH3PbI3 (reaction 2)Typically, the perovskite adopts a chemical formula denoted by The reported high–efficiency devices are mostly based on these two reactions163NH3PbIx3−Clx, a precursor solution containing PbCl2 and CH3NH3I with a molar ratio of 1:3 in dimethylformamide (DMF) was prepared (donated as “PbCl2 + 3CH3NH3I system”). The mixed solution was then spin–coated on TiO2 compact layer on an FTO–coated glass. The formation of final perovskite film was achieved by annealing at 100 oC for 45 min. X-ray diffraction (XRD) pattern shows typical (110) and (220) peaks centered at 14.1o and 28.4o  of CH3NH3Cl in the film accounts for negligible chlorine left in the final perovskite193, CH3NH2 or neutral CH3Cl, etc. To obtain a definite answer, we managed to separately collect the acidic or basic gases by filtering through acidic or basic media. Fourier transform infrared spectroscopy (FTIR) is used to characterize the vibrational modes of the collected gases in a gas cell. When the gas product was purged through NaOH powder, the spectrum  and 2 peaks cespectrum  shows pe145 cm−1 . To confin 2 min . It is aCH3NH3Cl . HoweverCH3NH3Cl  and lite10) and 2 peaks ceoC. The rationality behind this experiment is that HCl and CH3NH2 generated from the precursor film are able to enter into the upper pre-formed perovskite film and react with it. As a consequence, the surface of the perovskite film was observed to turn back to yellow. Furthermore, annealing of the yellow sample can again lead to the formation of the perovskite, indicating that the reaction between PbCl2 and CH3NH3I is reversible. In this case, the reaction depicted by (1) can be more precisely described by equation (3):Because of the generation of gaseous products, we also observed that if the reaction was conducted at a reduced pressure, the reaction would speed up because of the fast removal of the gaseous products. This is a typical characteristic of the solid-state reaction involving gaseous product generation. On the other hand, the reaction rate is greatly decreased when the reaction was performed in an enclosed system where the generated gas is retained in the system, indicating that the escape of gaseous product is a driving force for the reaction. To examine the reversibility of the reaction, we place a perovskite film facing a precursor film. The bottom precursor film was then heated up to 100 3NH2 favours the entropy increase and high temperature promotes the forward reaction.Since ΔG = ΔH − T ·ΔS , the reduction of Gibbs free energy induced by the entropy increase becomes increasingly significant as the temperature increases. Apparently, the release of HCl and CHin situ XRD to probe the crystal structure evolution–associated intermediate chemical reactions. The precursor film was firstly pre-dried at 60 oC. XRD measurement displays several sets of diffraction patterns .To explore the reaction pathways, we apply patterns . First, o and 31.37o can be assigned to the diffractions of CH3NH3PbCl3,2 and CH3NH3Cl has also happened along with the ion exchange as in reaction (5). There is also XRD diffraction peak at 14.02o corresponding to CH3NH3PbI3 observed at this stage, indicating the reaction (6).Second, the peaks at 15.53o, 16.64o, 28.42o and 18.03o cannot be assigned to any perovskite or lead halide; these peaks are presumably associated with the complex intermediate phase that composed of Pb halide and the organic species (Pb-complex)The diffractions at 11.38oC, the typical XRD diffraction pattern , (200), (210) and (300) plane with a cubic structure2 and CH3NH3PbCl3, accompanied by the growth of the diffraction peaks of CH3NH3PbI3  and (8) as indicated in o, 16.64o, and 18.03o gradually disappears as a result of the consumption of Pb for the formation of perovskite at elevated temperature (Another notable observation from the XRD pattern is that the (100) and (200) diffraction peaks downshift continuously during the annealing at 100 oC , which cperature .Ea) of the reaction. To obtain Ea, we first of all explore a parameter that can quantify the reaction rate. Considering the nature of the solid-state reaction, the gas release speed is associated with the reaction rate. If we assume that the overall reaction is described by reaction (9) below, a complete transformation to the perovskite leads to a weight loss of 17.9% as the gaseous products escape. This complete transformation to a pure-iodide based perovskite is reasonable since prolonged heating often results in undetectable chlorine content, both observed in our investigations and in the literatureThe kinetic factor that influences the reaction rate is usually reflected by the activation energy  of reaction (9), the precursor solution was pre-dried at 80  mixture . Subseques 15.2% , which iRr) against time (t) to determine the order of the reaction. The constant reaction rate as a function of time (Rr = k (k is the rate constant of the forward reaction). The natural logarithm form of Arrhenius equation can be expressed by formula (10).We thus plot the reaction rate  −1/T plot , (5) and (6) are nearly instantaneously occurred after drying at 60 oC. The calculated Ea represents required energy for the conversion of Pb-complex, CH3NH3PbCl3 (reaction d), CH3NH3PbI3−xClx (reaction e), to CH3NH3PbI3.where 1/T plot  we can c3NH3PbI3 displays zero-order reaction characteristics.Since the conversion towards final all-iodide perovskite is reversible and involves gaseous products generation, the increase in reactant would increase the distance for the gases diffusion out of the film. Furthermore, the increased diffusion length would increase the back reaction. An overall result is that the reaction rate did not show increment along with the increase of reactant amount. This is the reason why the formation of CH2 and CH3NH3I as the precursors, the reaction between PbI2 and CH3NH3I is conducted in a 1:1 ratio to generate CH3NH3PbI3 without chlorine doping (denoted as “PbI2 + CH3NH3I system”). The electronic properties of the two kinds of perovskites and their film formability are quite different2622 and CH3NH3I at 60 oC, the yielded light yellow powder was heated at elevated temperature to facilitate the reaction. The weight loss of the reaction was found to be 10.2% (Different from the perovskite synthesis using PbClbe 10.2% . From thRr (at constant temperature of 110 oC) against t to determine the order of the reaction  – 1/T plot (2 + 3CH3NH3I system (69 kJ mol−1). For the analysis of Ea, we characterized the dried mixtures of the two systems. It was found that the “PbCl2 + 3CH3NH3I” system forms poor crystallized product while the “PbI2 + CH3NH3I” system forms better crystallized material according the XRD characterization , the PCE can reach 12.26%, with a short-circuit current density (Jsc) of 19.66 mA cm−2, an open-circuit voltage (Voc) of 1.00 V, and a fill factor (FF) of 0.62. The average PCEs and standard deviations based on over twenty devices of each set are summarized in 2 and CH3NH3I as the precursors (device 2) leads to a PCE of 4.29%, with Jsc of 13.14 mA cm−2, Voc of 0.82 V and FF of 0.40. When using the mixture of lead precursors with CH3NH3I:PbCl2:PbI2 of 1.5:0.25:0.75 (device 3), 2.0:0.5:0.5 (device 4) and 2.5:0.75:0.25 (device 5), the PCEs are 13.78%, 15.00% and 13.05%, respectively. In the device fabrication, we found that to maintain 1:3 ratio between PbCl2 and CH3NH3I and 1:1 ratio between PbI2 and CH3NH3I in the precursor is crucial for the high–quality photoactive films.The photocurrent density-voltage responses of the devices  and the 3, 4, 5) with films prepared by using mixed PbCl2 and PbI2 sources are larger than that of the two devices fabricated using only PbCl2 or PbI2 as the lead precursor. The FF for the optimal device reaches 0.72 (device 4). FF is a parameter that reflects the quality of the device and is associated with the total resistance of the devices. The FFs of the devices based on the planar heterojunction , 1:0:1 , 1.5:0.25:0.75 (device 3), 2:0.5:0.5 (device 4) and 2.5:0.75:0.25 (device 5). The total concentration of lead salt in each solution was kept at 0.88 M.All materials were purchased from commercial suppliers and used as received unless stated otherwise. CH−1 was washed by sonication with deionized water, ethanol and acetone and then treated with oxygen plasma for two minutes. A compact layer of TiO2 was deposited on the FTO substrate by spin-coating the titanium precursor (0.24 M titanium isopropoxide and 0.12 M HCl in ethanol) at 5000 r.p.m. for 60 s following by calcination on a hotplate at 500 oC for 40 min. Subsequently, the perovskite solution was spin-coated on the cooled TiO2/FTO substrate in a nitrogen-filled glovebox at 3000 r.p.m. for 60 s. It was annealed on a hotplate at 100 oC for the reaction and crystallization of the perovskite. The optimized annealing time for the above-mentioned five precursors is 45 min, 10 min, 15 min, 23 min and 33 min, respectively. Then, the HTM, spiro-OMeTAD, was deposited by spin coating a solution  and 26.3 μL lithium-bis(trifluoromethanesulfonyl)imide (Li-TSFI) stock solution (520 mg mL-1 in acetonitrile) in 1 mL chlorobenzene) at 5000 r.p.m. for 60 s. After oxidizing the HTM layer in air for 15 h, the cell was completed by thermally evaporating a 100 nm-thick silver layer.FTO-coated glass with sheet resistance of 14 Ω sq−1. 25 μL perovksite solution containing CH3NH3I and PbCl2 of a molar concentration of 2.64 M and 0.88 M (mass percentage 42%) was held at 80 oC in a ceramic crucible for 150 min until the weight kept unchanged. A weight loss of 56% meant that DMF nearly totally evaporated. Then the remaining solid was heated from 80 oC to 180 oC at a rate of 5 oC min−1 during which CH3NH3PbIx3-Clx formed in the reaction  solution was held at 60 oC until the solvent DMF was totally evaporated. Mass loss of 60% meant that one DMF molecule is possibly binding with one Pb atom in the remaining solid. Then the yielded light yellow solid was heated from 60 oC to 110 oC and held at 110 oC till the complete reaction , the readily spin-coated perovskite film on TiO2/FTO substrate was transferred from a glovebox into an XRD characterization cell which was filled with nitrogen. The temperature of the characterization cell was held at 60 oC at first and then increased to 100 oC and kept for 50 min until the complete formation of CH3NH3PbI3. During this annealing stage, in situ XRD characterization  was performed from 10 degree to 50 degree (2θ) at a speed of 5 degree/min at a condition of 40 kV and 80 mA. The characterization process was repeated every ten minutes to monitor the phase change during the annealing stage.For the −1 from 10 degree to 70 degree (2θ)  .−1. Two probes mounted on the micropositioners were put in touch with the FTO and the silver electrodes, respectively. A scanning voltage from −0.2 V to 1.2 V was applied across the two electrodes at a step of 0.05 V and the corresponding current was recorded. All the cells had an active area of 0.12 cm2 defined by the silver electrode.The J-V characterization of the solar cells was conducted in a nitrogen-filled glovebox under the luminescence of AM 1.5 G solar-simulated light with an intensity of 100 mW cmThe incident photon current efficiency (IPCE) of the cells was characterized in air under the luminescence of monochromatic light from 300 nm to 800 nm in a DC mode without bias light. The output current of the cell was record at each wavelength. With the incident light powder known, the conversion efficiency from incident photons to the output charges was thus calculated for the whole spectrum.Rr) of the PbCl2 + 3CH3NH3I system was derived by taking a derivative on the weight of the reactant as a function of time at an isothermal temperature of 150 oC. The calculated reaction rate was 0.004 mg min−1 and gradually decreased to 0.003 mg min−1 after 150 min which could be considered as constant. It should be noted that the amount of the reactant is significantly larger than that in the device fabrication, so the required time for completing the reaction becomes much longer. On the other hand, with the generation of product in a solid-state reaction, the diffusion time of reactants becomes longer and longer, the apparent reaction rate thus becomes slower and slower.The reaction rate  – 1/T plot (k represents the reaction rate constant and T is thermodynamic temperature). Since the reaction between CH3NH3I and PbCl2 is a zero order one, then we can write A is the pre-exponential factor and R is the universal gas constant) based on Arrhenius equation. The reaction rate was calculated within a temperature range from 120 oC to 150 oC. For the reaction of the PbI2 + CH3NH3I system, we can write that W respresents the weight of the reactant since this is a first-order reaction. The data for plotting ln(k) – 1/T is in the temperature range between 70 oC and 100 oC.The activation energy of the above two reaction systems was calculated from the fitted slope of ln.


This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age  was associated with a greater relative abundance of Mycobacterium avium subsp. avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, including Mycobacterium abscessus and Mycobacterium chelonae. Overall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM.Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTM  rpoB genes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). The extraction procedure recovered, on average, eight times as much NTM DNA and three times as much total DNA from DW as two widely used commercial DNA extraction kits. The combined DNA extraction and sequencing approach allowed high-throughput screening of DW samples to identify NTM, revealing that the relative abundance of Mycobacterium avium subsp. avium increased with water age. Furthermore, the two-step barcoding approach developed as part of the PacBio sequencing method makes this procedure highly adaptable, allowing it to be used for other target genes and species.An extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTM  Management of the microbial quality of drinking water (DW) is aimed primarily at minimizing illness caused by waterborne pathogens. The increasing prevalence of infections due to nontuberculous mycobacteria (NTM) –4, combi–5–Mycobacterium avium and Mycobacterium abscessus) (Mycobacterium frederiksbergense and Mycobacterium aurum) (Only a few of the >150 NTM species that have been described  cause inscessus) , 10, 13,m aurum) , 15. MolrpoB and hsp65, is required to differentiate NTM species, as even full-length 16S rRNA gene sequences do not provide the resolution needed for discrimination to the species and subspecies levels  with low error rates. The high guanine-cytosine content of NTM genomes  providesIn this study, we sought to determine if single-molecule real-time sequencing with the PacBio platform  could overcome these obstacles. This platform achieves long read lengths  with low error rates when used in conjunction with the circular consensus sequencing (CCS) approach, making it possible to distinguish single nucleotide polymorphisms (SNPs) and allowing, in some cases, strain level differentiation –29. Comb–P < 0.001), as determined by measuring NTM abundances by quantitative PCR (qPCR) targeting the atpE gene  and NTM abundances (P = 0.045) . Likewise, there was no significant difference in the total bacterial, NTM, and Pseudomonas abundances obtained with increased bead-beating times (from 45 s to 2 min to 5 min) by either PC1 or PC2 (Two phenol-chloroform methods (designated PC1 and PC2) and two commercially available DNA extraction spin kits (FastDNA and Maxwell)  were comtpE gene . Howevermparable . PC2 emp= 0.045)  than the= 0.045)   with 10.1128/mBio.02354-17.1TEXT S1 TEXT S1, PDF file, 0.04 MB.Extraction efficiency calculation. Download Copyright © 2018 Haig et al.2018Haig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the rpoB primers  and subspecies of Mycobacterium fortuitum and Mycobacterium mucogenicum , were correctly identified as M. avium subsp. avium and M. chelonae . The DS water residence times of these homes varied from 11 to 113 h according to the city’s hydraulic model. Homes were divided into groups near (<24-h DS residence time) and far from (>24-h DS residence time) the DW treatment plant, corresponding to low and high water ages , respectively.rpoB gene sequences per sample. The number of NTM OTUs present in DW samples ranged from 4 to 10 (see M. abscessus) and slowly  growing species and including three distinct strains of M. chelonae and two strains of M. mucogenicum (P = 0.057), as measured by using the Shannon index   NTM o 10 see . Overallogenicum . There wndex see . DW samp ± 0.09) . M. aviu10.1128/mBio.02354-17.3TEXT S3 TEXT S3, PDF file, 0.05 MB.Combined qPCR and PacBio data. Download Copyright © 2018 Haig et al.2018Haig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.001), with water age explaining 81% of the difference (P = 0.03). The residual monochloramine concentration (C) multiplied by water age , referred to collectively as Ct, was not a significant parameter in the multivariate model explaining NTM community structure. However, a greater Ct was significantly associated with a greater NTM qPCR abundance (P < 0.001), with the average concentration across all samples corresponding to 6.47 × 106 ± 1.03 × 106 NTM atpE gene copies/liter. Further, additional multivariate analysis using estimated M. avium subsp. avium concentrations, obtained by combining M. avium subsp. avium relative abundances determined by the PacBio sequencing method with NTM qPCR concentrations .Permutational multivariate analysis of variance revealed that the NTM community structures of the two water age groups were significantly different from each other  of NTM species and strains in biomass from 15 DW samples collected from premise plumbing. Concentrations were calculated by combining qPCR and PacBio data. Water age represents the total of water residence time in the DS and the stagnation time in premise plumbing. Download Copyright © 2018 Haig et al.2018Haig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the DNA extraction methods impact the abundance and types of microorganisms recovered from complex environmental and biological samples owing to physiological differences among microorganisms and differing extraction efficiencies , 32. GivEukarya, which have complex cell walls, making them difficult to lyse  by the PC2 method was 78.6%, considerably higher than values reported in another study, in which the extraction efficiency did not exceed 43.3% . Hence, fewer sequences needed to be discarded, making this new methodology more economical and effective. Application of the sequencing approach to DW samples revealed 13 NTM species, 5 of which are considered clinically relevant  than in this study  may have been sufficient to result in NTM infections, as previously demonstrated through dose-response models  (Combining relative abundances of NTM determined by the PacBio sequencing method with NTM qPCR concentrations see  suggests102 CFU) . Since t102 CFU) .M. avium subsp. avium than DW from homes closer to the treatment plant. If supported by future, larger-scale studies, the factors contributing to this intriguing finding can be elucidated. This will, in turn, allow for better estimates of the health risks posed by NTM in DW and will provide a foundation for studies to mitigate this risk.Methods are needed that yield high NTM DNA levels during extraction from low-biomass samples and recover relatively large, phylogenetically informative segments of NTM DNA to advance our understanding of the potential health risks posed by NTM in DW systems. Our use of a DNA extraction method that employs enhanced physical disruption and chemical lysis of NTM cells resulted in considerably greater yields of NTM DNA from DW samples than those obtained with commonly used commercial DNA extraction kits. The development of a two-step barcoding procedure with PacBio sequencing provided a high-throughput method to detect and differentiate NTM present in DW. Applying these methods to DW samples collected from homes receiving municipally treated DW suggests that water age impacts NTM distribution in DW. Most notably, our findings indicate that DW from homes with DS residence times of >24 h had higher concentrations of From October 2015 to February 2016, 1-liter cold water samples were collected from kitchen faucets after at least 6 h of stagnation from 15 homes serviced by the Ann Arbor, MI, DW treatment plant. This first 1-liter sample represents the cold water in each home’s plumbing; the minimum premise plumbing volume for the homes sampled was 3.5 liters. The DW treatment plant obtains raw water from the Huron River (80 to 85%) and from groundwater wells (15 to 20%) and provides lime softening, coagulation, flocculation, sedimentation, ozonation, filtration, and chloramination . The homC) multiplied by water age , referred to collectively as Ct, was calculated for each sample.The temperature, pH, and total and free chlorine concentrations of all samples were measured on site. The average (±SD) water temperature and pH of the 15 samples were 20.4°C ± 1.9°C and 8.6 ± 0.6, respectively. Total and free chlorine concentrations were measured with a DR900 spectrophotometer . The combined chlorine (monochloramine) concentration was estimated by subtracting the free chlorine concentration from the total chlorine concentration. DS residence times for each home sampled were estimated by performing 168 h (7 days) of simulations in EPANET  for eachBiomass was filtered from each sample with a 0.2-μm polycarbonate filter  and stored at −80°C.Pseudomonas, and total bacteria as described below. Subsequently, DNA was extracted from 15 DW samples collected from homes and one mock community by PC2, the phenol-chloroform-based extraction method with 0.09% SDS and 5 min of bead beating.Four DNA extraction methods were tested in triplicate with 1-liter samples of DW from a composite sample of 30 liters of cold water collected from the Environmental and Water Resources Engineering Building at the University of Michigan (Ann Arbor). The four methods tested included the commercially available FastSpin kit for soil  and Maxwell LEV Blood DNA kit  and two variations of a standard phenol-chloroform method (PC1 and PC2) with a modified version of the universal nucleic acid extraction buffer  Table 1Table 1. 10.1128/mBio.02354-17.5TEXT S5 TEXT S5, PDF file, 0.1 MB.Detailed extraction procedure used in this study. Download Copyright © 2018 Haig et al.2018Haig et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli ATCC 15597 at 1.6 × 107 ± 7.1 × 106 CFU/liter or M. abscessus ATCC 19977 at 1.4 × 107 ± 1.1 × 107 CFU/liter. The cultures were grown in accordance with ATCC standard procedures. Resulting DNA yields were measured with the Qubit Fluorometer . Final extraction efficiencies were calculated by comparing the measured results to the extraction-independent total cell counts determined by staining with 4′,6-diamidino-2-phenylindole  and visualization with a Zeiss Axio Observer D1 fluorescence microscope . Theoretical DNA yields from E. coli and M. abscessus cells were calculated as outlined by Dolezel et al. . PCR conditions for NTM and total 16S rRNA gene assays consisted of 40 cycles, whereas the Pseudomonas assay was performed with 35 cycles. Melting curve analysis of the PCR products was conducted following each assay to confirm that the fluorescence signal originated from specific PCR products and not from primer dimers or other artifacts. For all qPCR assays, a linear relationship between the log of the standards’ DNA copy number and the calculated threshold cycle value across the specified concentration range was confirmed . Amplification efficiencies, calculated by the method described by Pfaffl . A phylogenetic tree was plotted with FigTree v1.4.2.MrBayes version 3.2  was usedM. avium subsp. avium (DJO-44271) and 5% M. chelonae (19237) by a two-step PCR procedure similar to that described in reference rpoB genes were amplified with the newly designed rpoB primers , we employed BLAST (Phylogeny of OTUs in the noncontrol samples was established on the basis of parameters obtained by testing a UPARSE analysis workflow  with theed BLAST  to identrpoB database was generated by downloading all 70,507 currently available bacterial rpoB sequences from GenBank (rpoB sequences. By using the aforementioned workflow, classifications were assigned to OTUs from the experimental samples by using global alignments with USEARCH and an identity threshold of >99.8%.The custom-made  GenBank , identif GenBank . Five yeMycobacterium, Pseudomonas, and total bacterial gene copy numbers obtained by different extraction methods were identified by Wilcoxon tests. Significant differences in the NTM community structure were determined by nonparametric multiple analysis of variance. All statistical analyses were performed with the R statistical software (P values of <0.05.Significant differences between the total DNA yields and software , with st


Mage = 13.7, SD = 0.53; 53.9% girls) and 845 9th grade students at T2  from the initial sample]. Besides direct effects, three cross-lagged over-time paths were found to be significant: students’ perception of peers as positive motivators (PPMs) at the beginning of 8th grade (T1) positively predicts their behavioral school engagement at the end of 9th grade (T2), as well as emotional school engagement at the beginning of 8th grade positively predicts students’ perception of PPMs 1.5 years later. Furthermore, behavioral school engagement at T1 functions as a predictor of a student’s school self-concept at T2.Existing literature evidences the association between adolescents’ school self-concept and engagement, both concepts being related to students’ perception of teachers and peers as motivators. However, few longitudinal studies explore the interplay of these factors. The present study aims to close this gap, applying latent cross-lagged panel design to two-wave data from German adolescent students [1088 8th grade students at T1 ( Figure 1).School engagement is an important factor in a student’s school career, as high engagement levels can enhance academic motivation and achievement , whereasHowever, until today it is not clear whether these relations are in fact reciprocal within and over time through adolescence, or whether there is another clear  order. This study aims to close this research gap and examine the within- and over-time associations of these variables from early to middle adolescence to gain a better understanding of the processes that accompany the trend of adolescents’ decline of engagement in school context. The findings might indicate potential starting points for prevention or intervention strategies to protect students from this downward trend.School engagement is defined as a complex and multidimensional construct , comprisThe self-system model of motivational development  suggestsAs mentioned-above, the Bioecological Theory , 1979, LPositive and motivating relations to peers are associated with an increase in both emotional and behavioral engagement at school . LongituAccording to the Bioecological Model of In sum, the existing body of research suggests that (a) school self-concept, emotional and behavioral school engagement and teachers and peers as positive motivators might associate with one another and (b) that these associations might be bidirectional according to the ideas of the Bioecological Theory and Developmental Contextualism and (c) that they might exist not only within-time but also over-time e.g., .Accordingly, this study follows a cross-lagged panel research design to evaluate the interplay of school self-concept, socio-motivational relations with teachers and peers, and emotional and behavioral school engagement within- and over-time from the beginning of 8th grade to the end of 9th grade in secondary school context. Specifically, it was hypothesized that higher levels of school self-concept and a more positive perception of peers and TPMs would be concurrently and longitudinally related to higher levels of emotional and behavioral school engagement.Mage = 13.7, SD = 0.53; 53.9% girls)] at Time 1 and remaining 845 students  from the initial sample at Time 2 (1.5 years later at the end of 9th grade). This age group was chosen for two reasons: (1) As the school transition to secondary school occurs in Germany in 7th grade, students tend to struggle with the associated intra- and interindividual changes still at the beginning of 8th grade  Furth grade  reachingth grade . The parSchool Engagement measures are based on the Engagement/ Disaffection Scales developed by 1) and the ESE Scale   were comprised of six items each. Although the Cronbach alpha value for the ESE items at T2 was not as high as for the other subscales, parcels can be built according to School Self-Concept (SSC) was addressed by a subscale of SESSKO scales developed by Teachers and Peers as Positive Motivators was assessed using Relationship and Motivation (REMO) Scales (The perception of ) Scales ; (1) TPM2-difference test was estimated using the Satorra–Bentler scaling correction factor , Root Mean Square Error of Approximation (RMSEA), Comparative Fit Index (CFI), Tucker-Lewis Index (TLI) and Standardized Root Mean Square Residuals (SRMR). Respective CFI and TLI values above 0.95 and RMSEA and SRMR values up to 0.08 indicating an acceptable fit of the model. Due to the fact that missing data was completely at random (MCAR) as shown in 2 = 117.35; df = 101; p > 0.05), missing data were handled using full-information maximum likelihood estimation (FIML).We used the TYPE = COMPLEX function in Mplus to consider the classroom nesting of the data, because it supplies corrected standard errors and chi-square values regarding the nested structure of the data (1088 students in 71 school classes) . Model fTable 1. Results from an unconditional latent change model (LCM) , ESE  and PPM . In turn, there was neither a significant mean decrease/increase in TPM  nor in SSC .Bivariate correlations and descriptive statistics are reported in el (LCM)  that inc2(125) = 358.57, p < 0.001; CFI = 0.96, TLI = 0.95, RMSEA = 0.04 (0.04–0.05); SRMR = 0.03].Before conducting the cross-lagged panel design, a confirmatory factor analysis (CFA) was run. The CFA showed a good model fit [χTable 2). Strong measurement invariance over time has been found, supporting the assumption that the constructs remained stable over time and therefore, flavoring the use of cross-lagged panel design  specified an unconditional model  without equality constrains; (2) specified factor loadings as invariant over time ; and (3) set loadings and item intercepts invariant over time   showed a good fit χ2(123) = 308.842, p < 0.001, CFI = 0.97, TLI = 0.96, RMSEA = 0.04 (0.03–0.05), SRMR = 0.03.The final cross-lagged panel design model  as well as the within-time associations between BSE and ESE  Furthermore, TPM is positively associated with BSE  and ESE . In turn, the association between PPM and BSE  and between PPM and ESE  was significant solely at T1. In addition, the association between SSC and BSE  as well as the association between SSC and ESE  was found to be positively significant. In contrast, there was no significant relation between SSC and PPM neither at T1 nor at T2. In turn, a positive significant association between SSC and TPM was found .The within-time associations between TPM and PPM are positively significant , ESE , TPM , PPM , SSC .The model evidenced positive direct effects of each variable from T1 to T2 supporting the stability of the constructs: BSE . Furthermore, ESE at T1 positively predicted PPM at T2 . In turn, SSC at T1 negatively predicted both ESE  and PPM at T2 .Students’ perception of PPM in early adolescence predicted BSE in middle adolescence  school engagement, whereas this role generally decreases in importance during middle adolescence. This finding is in accordance with research that showed that peers as secondary socialization instance are particularly important in early adolescence, when students turn away from family  and haveIn addition, there was neither a significant within-time association between PPMs and school self-concept at T1 nor at T2. This means that adolescents’ school self-concept is less sensitive to motivational support from peers compared to motivational support from teachers (see above). This may be due to the fact that teachers in their institutional role possess more opportunities to promote a student’s academic self-concept: TPMs may not only satisfy students’ affiliative needs, but also support the needs of competence and autonomy by clear set goals, well-articulated expectations, meaningful instructions and empowerment . In turnOverall, our two-wave cross-lagged panel design study showed that the within-time interplay between school self-concept, behavioral and emotional school engagement as well as students’ perception of peers and TPMs tend to be stronger in early adolescence at the beginning of 8th grade than in middle adolescence at the end of 9th grade. This might be explained by a growing need for autonomy from adults during middle adolescence  and incrIn particular, the role of PPMs seems to loose impact from early to middle adolescence: while PPMs were positively associated with both behavioral and emotional school engagement at T1 and positively associated over time with behavioral school engagement, there was no significant association of PPMs and behavioral school engagement at T2. However, there was a positive over-time association between emotional school engagement at 8th grade and students’ perceptions of PPMs at the end of 9th grade, not only supporting the existing findings that these two factors positively associate , but alsIn sum, our findings underline the relevance of Bronfenbrenner’s Bioecological Theory and Lerner’s Developmental Contextualism in the explored interplay in a short-term perspective, such as peers and teachers play differentiated roles for different aspects  at different times during adolescence and vice versa. In other words, the development of adolescent students’ school engagement is embedded in complex dynamics between a student’s sense of their own abilities in school and their motivational relationships with peers and teachers within-time, although no relation between the variables is bidirectional over-time. Therefore, fostering both students’ school self-concepts as well as their motivational relations with peers and teachers might benefit their emotional and behavioral school engagement and vice versa in the course of adolescence, whereas the beneficial effect is greater the earlier it starts.The current research evidences several theoretical and methodological limitations. First, although students’ subjective perceptions were at the heart of the study, self-report is often subject to criticism. However, some authors  claim thAt the same time, the current study evidences a number of strengths. Firstly, it focuses on absolute school self-concept, allowing practical interventions through school psychologists, as impact on only one aspect of school self-concept within school environment  might induce negative changes in the counterpart e.g., . Secondlhttp://bravors.brandenburg.de/verordnungen/wissuv_1998) with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ministry of Education, Youth and Sports of Brandenburg.This study was carried out in accordance with the recommendations of the guidelines of the Ministry of Education, Youth and Sports of Brandenburg (All authors agree to be accountable for the content of the work. OB did the statistical analyses and wrote the main part of the paper. DR assisted with the statistical analyses and reviewed the paper, contributed decisively to the discussion part and added comments on the manuscript throughout the process of manuscript development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MATa and MATα), the protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling pathways integrate at the promoter of the master regulatory transcription factor IME1 to control sporulation via nutrient availability , however, IME1 is repressed by transcription through the IME1 promoter of a long non-coding RNA called IRT1, which prevents this cell type from undergoing sporulation. Here, we investigated the role of nutrient signalling in mating-type control of IME1. We find that expression of IRT1, like IME1 itself, depends on nutrient availability and the activities of PKA and TORC1. IRT1 transcription is repressed when nutrients are ample and TORC1 and PKA are active. In contrast, inhibition of PKA and TORC1 is sufficient to recruit Rme1 to the IRT1 promoter and induce IRT1-mediated repression of IME1. Finally, we provide evidence that IRT1 and IME1 are co-repressed by the Tup1–Cyc8 complex when nutrients are ample. Thus, in cells with a single mating-type nutrient availability regulates mating-type repression of IME1 and sporulation. Our results indicate that there is a hierarchy between nutrient and mating-type signals in controlling the decision to enter sporulation.Cell fate decisions are controlled by multiple cell-intrinsic and -extrinsic factors. In budding yeast, the decision to enter gametogenesis or sporulation is dictated by nutrient availability and mating type. Recently, we showed that in diploid cells harbouring opposite mating types ( Cell fate decisions are regulated by a multitude of intrinsic and extrinsic signals. How multiple signals are integrated to make a binary cell fate choice is often not well understood.Budding yeast gametogenesis or sporulation is an ideal model system to study this problem. When the signal requirements to enter sporulation are met, cells induce a gene expression cascade to generate four haploid spores from a diploid cell , are active , represses the transcription of RME1 (Mitchell and Herskowitz MATa or MATα), however, exhibit high levels of Rme1 which in turn represses IME1 via an unusual mechanism . The mating-type locus (HMR) was used as a non-binding negative control. Primer sequences are available on request.Chromatin immunoprecipitation experiments were executed as described previously  that was described previously and the TORC1 inhibitor rapamycin  . Interestingly, the levels of IRT1 were significantly higher when tup1Δ cells were grown to high density (OD600 ~ 10). We conclude that Tup1–Cyc8 contributes to IRT1 repression.Having established that TORC1 and PKA control IME1, IRT1 (at least in part) is regulated by Tup1–Cyc8. How Tup1–Cyc8 represses both IRT1 and IME1 remains unclear. Promoter scanning showed that Tup1 binding peaked in the middle of the IME1 promoter , we propose that Tup1–Cyc8 establishes a repressive chromatin state at the IME1 promoter that spreads to the IRT1 promoter. Our observation that IRT1 expression was further induced during post-diauxic shift in tup1Δ cells suggests that Tup1–Cyc8 is not the sole repressor of IRT1 and other factors may also contribute. Another explanation is that the activator of IRT1, Rme1, is regulated by nutrient availability. It is known that Rme1 levels are cell cycle regulated, peaking during late M/early G1  as IME1. In addition, we provide evidence that IRT1 and IME1 are under control of the same repressor complex, Tup1–Cyc8. These findings have several implications. First, they show that mating-type control of IME1 is not active when nutrients are ample (Fig. IME1 and IRT1 could serve as a fail-safe mechanism for mating-type control of IME1. Indeed, it has been known that mating-type control of sporulation is essential for preventing haploid cells to enter meiosis, which would be lethal as two consecutive cell divisions would attempt the segregation of a haploid genome into four spores. If IRT1 and IME1 were under control of different signalling pathways and different transcriptional repressors, this could have led to a mis-regulation of mating-type control of IME1. Our observation that IME1 and IRT1 expressions are regulated by the same nutrient sensing pathways and by the same transcriptional repressor complex ensures that IRT1 is activated at the same time when the nutrient requirements for IME1 are met and vice versa. Overall, we propose that the hierarchy between nutrient and mating-type signals ensures that diploid, and not haploid, cells induce IME1 and enter sporulation.It has been known for decades that nutrient availability and mating type are key regulators of ple Fig. . In othe


Several congenital and acquired conditions may result in severe narrowing of the urethra in men, which represent an ongoing surgical challenge and a significant burden on both health and quality of life. In the field of urethral reconstruction, tissue engineering has emerged as a promising alternative to overcome some of the limitations associated with autologous tissue grafts. In this direction, preclinical as well as clinical studies, have shown that degradable scaffolds are able to restore the normal urethral architecture, supporting neo-vascularization and stratification of the tissue. While a wide variety of degradable biomaterials are under scrutiny, such as decellularized matrices, natural, and synthetic polymers, the search for scaffold materials that could fulfill the clinical performance requirements continues. In this article, we discuss the design requirements of the scaffold that appear to be crucial to better resemble the structural, physical, and biological properties of the native urethra and are expected to support an adequate recovery of the urethral function. In this context, we review the biological performance of the degradable polymers currently applied for urethral reconstruction and outline the perspectives on novel functional polymers, which could find application in the design of customized urethral constructs. The male urethra can be affected by several primary, as well as secondary conditions, including hypospadias, fistulas, trauma, and malignancy . In chilOverall, disorders of the male urethra may lead to a breach of the continuity of the urethral mucosa, which results in extravasation of urine, with the possibility of subsequent inflammation and, ultimately, stricture formation. The surgical management of urethral strictures differs according to the severity and length of the affected segment. While the basic approach for short segmental defects comprises resection and end-to-end anastomosis, long defects require open urethroplasty using autologous tissue grafts. Buccal mucosa is currently the most widely used graft material for urethroplasty ,8,9. HowUrethral tissue engineering has recently emerged as a viable alternative to overcome the issues associated with autologous grafts . In addiThe aim of this article is to present the rationale for different designs for urethral replacement, as well as the limitations of each design choice. Given the inability of grafts and non-degradable polymers to meet the clinical performance requirements, this review will focus on the ability of degradable polymeric scaffolds to mimic the native urethral structure, in an effort to restore complete urethral function. Toward this purpose, we initially describe the properties of the male urethra that dictate design requirements for a completely regenerated structure. Then, we review the preclinical and clinical results obtained with current biodegradable biomaterials, discussing their advantages and limitations. Finally, we discuss the perspectives on novel functional polymers, which may find application in the field of urethral tissue engineering.2O. The most distal and longest portion is the penile urethra, which is surrounded by the corpus spongiosum, a richly vascularized erectile tissue that provides support to the urethra. Histologically, the urethra is composed from three different layers: an inner mucosal epithelial lining, a submucosa comprising of fibroblasts, and a muscular layer  can explain the relation between the structure and function of the urethra. Studies have shown that the urethra possesses a nonlinear cross-sectional area pressure, meaning that the tissue is fairly deformable at low pressure (facilitating voiding), but less deformable at higher stress levels (protective against over distention) . It has Adding to the complexity of the picture, in hypospadias and urethral surgery, additional biomechanical and clinical problems may arise ,35,36. AAnother important point that has not received too much attention in the design of scaffolds for urological tissue engineering is the avoidance of urine leak, even when the urine is known to be highly cytotoxic. In the normal urethra, urothelium barrier function is maintained by three structures: uroplakin proteins in the apical cell membrane, tight junctions situated between the superficial umbrella cells, and urothelial GAGs and proteoglycans, covering the umbrella cells . FormatiInitially, non-degradable materials were assessed for urinary tract reconstruction, but they resulted into several complications such as calcification, fistulae ,45, chroThe appropriate synchronization between the biodegradation of the scaffold and cellular component growth is crucial for the success of a tissue-engineered implant. The scaffold should tolerate the mechanical forces sustained during handling, suturing, and normal patient activities . BiodegrDegradable polymers for urethral tissue engineering have been typically obtained from natural sources or by synthesis. Natural biomaterials include acellular matrices obtained from cadaveric or animal organs via enzymatic, physical, or chemical decellularization methods, or natural polymers, such as silk fibroin (SF). In general, natural biomaterials possess integrin-binding peptide sequences and surface topography that can facilitate cellular growth and differentiation, and accelerate the process of angiogenesis . SynthetAcellular matrices, such as small intestinal submucosa (SIS) and bladder acellular matrix (BAM), are by far the most frequent scaffold type assessed in vivo for urethral tissue repair . SIS is BAM is another example of a decellularized matrix, which has shown to successfully support regeneration of the urethra in vivo ,74,75,76Bombyx mori cocoons that holds promise for urethral tissue engineering applications [SF is a natural polymer derived from ications ,80. Tradications ,82. SF iications . In compications ,85. It wications . Xie et ications . As it was previously shown, non-degradable materials did not meet the clinical performance requirements. Despite the moderate success of degradable scaffolds in preclinical studies, the transition from bench-to-bedside remains a challenge. There are, however, some novel approaches and materials being investigated that might be able to meet the clinical performance requirements, alone or in combination with some of the other systems presented here. Natural and synthetic materials can be combined to produce hybrid biomaterials with desired properties for tissue engineering applications. Such properties include mechanical strength, porosity and cell affinity to attract cells, biocompatibility, and biodegradability for enabling replacement of ECM produced by resident cells. Current efforts in biodegradable polymer synthesis are centered on determination of appropriate biomaterials and synthesizing these materials with tailored properties for specific applications. Another important advancement within the present decade is the emergence of a fourth generation of “smart” biomimetic materials. These materials respond reversibly to temperature, ionic strength, pH, or light ,88. The Thermo-responsive polymers are the largest class of smart polymers. These are characterized with a reversibly alterable phase (or volume) transition that occurs in response to a change in temperature. The solubility of thermo-responsive polymers in aqueous solutions depend on the temperature. Above lower critical solution temperature (LCST) polymer chains are precipitated making it hydrophobic, and below LCST, polymer chains are completely hydrated, making it hydrophilic . The mosShape-memory polymers (SMPs) are another class of smart biomaterials. SMPs have been experimented in various vascular and bone tissue engineering/regenerative medicine studies ,97,98. FAnother subset of smart polymers that has shown promise for engineering electrically active tissues is the group of electroconductive polymers. Electroactive polyurethane polymers have shown great potential in bladder tissue engineering applications, when the aim is to regenerate muscular components and innervation ,103,104.Although several experiments have succeeded in regenerating the different urethral tissue layers  ,55,106 nIn the future, advances in understanding the physical and chemical factors responsible for urothelial and smooth muscle cell proliferation, migration, differentiation, and function may be exploited in the development of smart biomaterials, in which biologically active agents are incorporated into acellular natural or synthetic matrices. Not only interactions between urothelium and stroma, but also bi-directional interactions between tissue-engineered grafts and the host tissue environment, are important factors in achieving better outcomes in tissue remodeling. This concept has been termed “dynamic reciprocity” . With a The performance of the current treatments for long urethral strictures remains suboptimal due to the shortage of tissue sources, the significant donor site morbidity, or the inability to completely restore the structure and function of the host. While non-degradable polymers have not met the clinical performance requirements because of the potential risk of migration, encrustation, and ultimate narrowing, tissue-engineered solutions based on biodegradable polymers have emerged as potential alternatives for successful recovery, and therefore function, of the urethral structure. Biological scaffolds have shown utility to repair urethral defects, but they display relative mechanical inferiority and their degradability cannot be easily adjusted. On the other hand, synthetic scaffolds have highly controllable chemical, mechanical, and structural characteristics, but proper reproduction of the native extracellular environment remains a challenge. This suggests that combinations of natural and synthetic polymers could provide synergy with a controllable degradation rate, mechanical compliance, and supportive of formation of different tissue layers and vascularization. It is important to stress that a successful urethral scaffold should maintain clinical performance  following implantation, and synchronize with the preclinical performance requirements . This can potentially be achieved by controlled degradation matching regrowth of healthy tissues. Functional and smart polymeric scaffolds have the potential to better mimic the native tissue and may help tissue engineered solutions to meet the pre-clinical and clinical performance requirements in the future.


C. elegans, a neurotransmitter-sensing G protein-coupled receptor, TYRA-2, is required for avoidance responses to osas#9, an ascaroside pheromone that incorporates the neurotransmitter, octopamine. Neuronal ablation, cell-specific genetic rescue, and calcium imaging show that tyra-2 expression in the nociceptive neuron, ASH, is necessary and sufficient to induce osas#9 avoidance. Ectopic expression in the AWA neuron, which is generally associated with attractive responses, reverses the response to osas#9, resulting in attraction instead of avoidance behavior, confirming that TYRA-2 partakes in the sensing of osas#9. The TYRA-2/osas#9 signaling system represents an inter-organismal communication channel that evolved via co-option of a neurotransmitter and its cognate receptor.Biogenic amine neurotransmitters play a central role in metazoan biology, and both their chemical structures and cognate receptors are evolutionarily conserved. Their primary roles are in cell-to-cell signaling, as biogenic amines are not normally recruited for communication between separate individuals. Here, we show that in the nematode  Inter-organismal signaling is essential for animals to navigate and survive in their natural environment, yet is unclear how these chemical communication channels may have evolved. Here, authors show that TYRA-2, an endogenous tyramine/octopamine receptor, is required for the chemosensation of an octopamine-derived pheromone and that this signaling system represents an inter-organismal communication channel that evolved via co-option of a neurotransmitter and its cognate receptor Chemosensation, both ancient and ubiquitous across all kingdoms of life, underlies social responses mediated by chemical communication2. Social chemical communication requires both cell-to-cell and inter- organismal signaling. First, a chemical cue is released into the environment by one organism that is then detected by specific receptors in another organism. Upon sensation, inter-cellular signaling pathways, e.g., neurotransmitter signaling, are activated that ultimately coordinate a social response.Inter-organismal communication occurs in many forms across the animal kingdom, both within and between species3. The associated signaling pathways often rely on highly regulated biosyntheses, translocation (either by way of diffusion or through active transport), and perception by dedicated chemoreceptors. Many neurotransmitters are perceived via G protein-coupled receptors (GPCRs); in fact, there is a close relationship between GPCR diversification and neurotransmitter synthesis in shaping neuronal systems4. Notably, the most common neurotransmitters share similar behavioral functions across phyla. For example, serotonin is commonly involved in regulating food responses5. Other neurotransmitters, such as tyramine and octopamine, are only found in trace amounts in vertebrates, and act as adrenergic signaling compound in invertebrates6.Neurotransmitter monoamines, such as dopamine, serotonin, tyramine, and octopamine, serve diverse functions across kingdomsCaenorhabditis elegans offers many advantages for studying social chemical communication and neuronal signaling, namely the animal’s tractability, well-characterized nervous system, and robust social behavioral responses to pheromones7. C. elegans secretes a class of small molecules, the ascaroside pheromones, which serve diverse functions in inter-organismal chemical signaling9. As a core feature, these molecules include an ascarylose sugar attached to a fatty acid-derived side chain that can be optionally decorated with building blocks from other primary metabolic pathways9. Ascaroside production, and thus the profile of relayed chemical messages, is strongly dependent on the animal’s sex, life stage, environment, and physiological state12. Depending on their specific chemical structures and concentration, the effects of ascaroside signaling vary from social (e.g. attraction to icas#3) to developmental  and tyramine  for involvement in the osas#9 response  mutant, whereas osas#9 avoidance was largely unaffected in the other neurotransmitter receptor mutants , copper chloride (CuCl2), and glycerol. No defects were found in tyra-2 lof animals’ ability to respond aversively to these deterrents  that has been shown to bind tyramine with high affinity and, to a lesser extent, octopaminents Fig. . This ins#9 Fig. . Wild-tyons Fig. . Howeverons Fig. .tyra-2 transcript levels changed under starvation conditions using quantitative RT-qPCR. Starved worms exhibited a nearly two-fold increase in tyra-2 expression . We observed TYRA-2 expression in four sensory neurons: ASH, ASE, ASG, and ASI 38 or peptidegergic signaling (unc-31)39 . The worms expressing the transgene displayed sub-cellular localization in the ASH sensory cilia  elicited by an odorant are specified by the olfactory neuron in which the receptor is present, rather than by the olfactory receptor itself42. We asked whether driving TYRA-2 receptor expression in other sensory neurons will drive behavioral response to osas#9. For this purpose, we ablated the ASH neurons in the pnhr-79::tyra-2 strain, in which tyra-2 is expressed in the ASH and ADL neurons 47. We tested mutants for each of those eight Gα subunits for their response to osas#9 , their translocation (either by way of diffusion or through active transport), and perception by dedicated receptors60. This mode of signaling is strikingly similar to pheromone-mediated communication systems, which rely on highly specific production and perception of small molecule ligands for inter-organismal signaling61. During evolution, it stands to reason that some machinery from inter-cellular signaling would also be utilized for inter-organismal signaling. Co-option of such signaling systems has been observed in both invertebrates (C. elegans), where a nicotinic acetylcholine receptor senses choline62, and vertebrates (such as mice and zebrafish), where some metabotropic neurotransmitter receptors act as sensors to detect amino acids in the environment65.Exactly how key innovations in metazoan signaling complexity evolved from pre-existing machineries remains to be elucidatedC. elegans , and a related receptor (TYRA-2) for mediating starvation-dependent dispersal in ans Fig. , suggest66. The avoidance response was characterized a reversal if the behavior consisted of at least one half of a complete “head swing” followed by a change in direction of at least 90° from the original vector. For quantitative analysis, an avoidance response was scored as “1” and no response as “0”. The avoidance index was calculated by dividing the number of avoidance responses by the total number of trials. Each trial was done concurrently with osas#9 and a solvent control.The tail end of a forward moving animal was subjected to a small drop (~5 nl) of solution, delivered through a hand-pulled 10 μl glass capillary tube. The solution, upon contact, was drawn up to the amphid sensory neurons via capillary action. In response, the animal either continued its forward motion (scored as “no avoidance response”), or displayed an avoidance response within 4 sControl animals and strains containing transgenes in various genetic backgrounds were prepared using common M9 buffer to wash and transfer a plate of animals to a microcentrifuge tube where the organisms are allowed to settle. The supernatant was removed and the animals were resuspended and allowed to settle again. The supernatant was again removed and the animals transferred to an unseeded plate. After 1 h, young adult animals were subjected to the solvent control and chemical of interest at random, with no animal receiving more than one drop of the same solution. Refed animals were transferred to a seeded plate with M9 buffer, and after the allotted time, transferred to an unseeded plate and tested after 10 minutes.66.Ablated and extrachromosomal transgenic animals and controls were gently passed onto an unseeded plate and allowed to crawl around. They were then again gently passed to another unseeded plate to minimalize bacterial transfer. Ablated animals were tested three times with the solvent control and solution of interest with 2 min intervals between dropstyra-2 rescue and misexpression plasmids were generated using MultiSite Gateway Pro Technology and injected into strain FX01846 tyra-2(tm1846) with co-injection marker pelt-2;mCherry by Knudra Transgenics. The promoter attB inserts were generated using PCR and genomic DNA or a plasmid. The tyra-2 insert was isolated from genomic DNA using attB5ggcttatccgttgtggagaa and attB2ttggcccttccttttctctt. PDONR221 p1-p5r and PDONR221 P5-P2 donor vectors were used with attB inserts. The resultant entry clones were used with the destination vector pLR305 and pLR306.odr-10 promoter was isolated from genomic DNA using primers attB1ctcgctaaccactcggtcat and attB5rgtcaactagggtaatccacaattc. Entry clones were used with destination vector pLR305 resulting in podr-10::tyra-2:: RFP and co-injected with pelt-2::mCherry into FX01846.For AWA expression, a 1.2-kb nhr-79 promoter was isolated from genomic DNA using primers attB1gtgcaatgcatggaaaattg and attB5ratacacttcccacgcaccat. Entry clones were used with destination vector pLR306 resulting in pnhr-79::tyra-2::RFP and co-injected with pelt-2::mCherry into FX01846.For ASH expression, a 3-kb nhr-79 promoter was isolated from genomic DNA using primers attB1gtgcaatgcatggaaaattg and attB5ratacacttcccacgcaccat. gpa-6 was isolated from genomic DNA using primers attB5 cgtctctttcgtttcaggtgtat and attB2 tattttcaaagcgaaacaaaaa. Entry clones were used with destination vector pLR304 resulting in pnhr-79::gpa-6::RFP and co-injected with punc-122::RFP into NL1146.For ASH expression, a 3-kb tyra-2::GFP fusions were created by PCR fusion using the following primers to isolate 2 kb ptyra-2 with its entire genomic locus from genomic DNA: A) atgttttcacaagtttcaccaca, A nested) ttcacaagtttcaccacattacaa, and B with overhang) AGTCGACCTGCAGGCATGCAAGCT gacacgagaagttgagctgggtttc. GFP primers as described in WormBook67. The construct was then co-injected with pelt-2::mCherry into both N2 and FX01846.gpa-6::RFP was generated by adding the restriction sites, AgeI and KpnI, to isolate 4 kb pgpa-6 and the entire gpa-6 locus from genomic DNA using primers: acatctggtacccctcaatttcccacgatct and acatctaccggtctcatgtaatccagcagacc. RFP::unc-54, ori, and AMPr was isolated from punc-122::RFP plasmid by PCR addition of the restriction sites AgeI and KpnI with primers: acatctaccggt ATGGTGCGCTCCTCCAAG and ttaataggtaccTGGTCATAGCTGTTTCCTGTG. After digestion and ligation, the clone was injected into N2 with co-injection marker punc-122::GFP.(See Supplementary Tables 13.ascr#3 and osas#9 were synthesized as previously describedhttps://www.sourcebioscience.com/products/life-sciences-research/clones/rnai-resources/c-elegans-rnai-collection-ahringer/). The RNAi clones  originated from the Vidal Library68, were generously provided by the Ambros Lab at UMASS Medical School. We observed that RNAi worked best when animals were cultured at 15 °C. We used VH624 (nre-1(hd20);lin-15B(hd126)) for the RNAi studies, as it has been previously shown to be sensitive to neuronal RNAi28.RNAi knockdown experiments were performed by following the RNAi feeding protocol found at Source Bioscience  was calculated by dividing the ΔF value of each frame by F0. F0 was calculated as the average ΔF of 10 frames prior to stimulus exposure37. ΔF/F0 (%) was calculated by subtracting 1 from ΔF/F0 and multiplying 100%; these calculations were then plotted over the duration of the experiment.Calcium imaging was perfomed by using a modified olfactory chip described in Reilly et al.71. cDNA was subsequently synthesized using the Maxima H Minus First Strand cDNA Synthesis Kit. iTaq Universal SYBR Green Supermix was used for amplification with the Applied Biosystem 7500 Real Time system. Primer efficiency was determined to be 97.4% for tyra-2 primers  and 101.8% for the reference gene ama-1  using the equation 10^(-1/slope)-1. Technical replicates with large standard deviations and trials with a Ct within 5 cycles of the negative control (no reverse transcriptase used in prep) were removed from analyses.RNA was isolated from individual animals, either freshly removed from food or after four hours of starvation using Proteinase K buffer: Five animals were gently transferred to a 35-mm plate and filmed for 20 minutes. Movies were generated using the Wormtracker system by MBF Bioscience. Movies were then analyzed and average speed was computed using software WormLab4.1 .Speed: reversals were analyzed and measured using Wormlab (MBF Bioscience) from movies recorded for the holding assay between minute one and two as it was when the divergence was first seen in distance between strains in the holding assay.Reversals72. 10 animals were placed in the center of a 35-mm plate, equidistant from two spots, one containing 1 µl of solvent control and the other 1 µl of 10-2 diacetyl. Both spots contained sodium azide for anesthetizing animals that entered the region. After 45 minutes, the chemotaxis index was calculated by subtracting the number of animals in the solvent control from the number of animals in the solution of interest and divided by the total number of animals.Diacetyl chemotaxis assays were carried out with slight modificationsE. coli OP50 liquid culture was spread onto a separate NGM assay plates. These plates were allowed to dry at 25 °C without a lid for one hour. After an hour of incubation, 4 µl of either solvent control or 10 pM osas#9 was pipetted onto the agar within the center circle outlined on the template. Ten animals were gently passed into the center circle and their movement was recorded. At 1-minute intervals, the distance the animals traveled from the origin was measured using ImageJ.The leaving assay consisted of the use of 60 mm culture plates containing standard NGM agar. A transparency template that included a 6-mm diameter circle in the center was attached to the underside of the NGM plate. One hour before running the assay, young adult animals were passed on to an unseeded plate and allowed to starve for one hour. 100 µl of t-test was used. All tests were two-tailed. When comparing different strains/conditions, normalized values of osas#9 avoidance index response relative to the respective solvent control were used. This was done to account for any differences in baseline response to solvent control for the respective genotypes, laser ablations, or physiological conditions. When normalizing fold change of osas#9 response to solvent control response for the avoidance assay within a strain/condition, data was first log transformed so a fold change could still be calculated for control plates that had a “0” value. For avoidance assays, statistical groups were based on the number of plates assayed, not the number of drops/animals. For calcium imaging, averages were calculated by obtaining the max peak value before and during exposure to the chemical of interest for each trial.Statistical tests were run using Graphpad Prism. For all figures, when comparing multiple groups, one factor ANOVAs were performed, followed by Sidak’s multiple comparison test. When only two groups were compared, a Student’s p-value as asterisks, but represent the difference between osas#9 avoidance of a strain/conditions in comparison to wild type, with the exception of panel 5H, which shows the difference between response of all strains/conditions and the reprogrammed AWA::tyra-2For all figures, asterisks depict compared osas#9 avoidance to respective solvent control within groups. ‘ + ’ signs represent same Further information on research design is available in the Supplementary InformationPeer Review FileReporting summarySupplementary Movie 1Source Data